Hyaluronan, a linear glycosaminoglycan comprising D-N-acetylglucosamine and D-glucuronic acid, is the main component of the extracellular matrix. Its influence on cell proliferation, migration, inflammation, signalling, and other functions, depends heavily on its molecular weight and chemical modification. Unsaturated HA oligosaccharides are available in defined length and purity. Their potential therapeutic utility can be further improved by chemical modification, e. g., reduction. No synthesis of such modified oligosaccharides, either stepwise or by hyaluronan cleavage, has been reported yet. Here we show a three-step synthesis (esterification, depolymerization and reduction) of unsaturated even numbered hyaluronan oligosaccharides with carboxylates and the reducing terminus reduced to an alcohol. Particular oligosaccharides were synthesised. The modified oligosaccharides are not cleaved by mammalian or bacterial hyaluronidase and do not affect the growth of mouse and human fibroblasts. Further, MTT and NRU viability tests showed that they inhibit the growth of human colon carcinoma cells HT-29 by 20-50 % in concentrations 500-1000 µg/mL. Interestingly, this effect takes place regardless of CD44 receptor expression and was not observed with unmodified HA oligosaccharides. These compounds could serve as enzymatically stable building blocks for biologically active substances.
Cell Proliferation , Cytostatic Agents , Hyaluronic Acid , Hyaluronoglucosaminidase , Oligosaccharides , Hyaluronic Acid/chemistry , Hyaluronic Acid/pharmacology , Humans , Oligosaccharides/chemistry , Oligosaccharides/pharmacology , Animals , Mice , Cell Proliferation/drug effects , Hyaluronoglucosaminidase/metabolism , Hyaluronoglucosaminidase/antagonists & inhibitors , Cytostatic Agents/pharmacology , Cytostatic Agents/chemistry , Cytostatic Agents/chemical synthesis , HT29 Cells , Hyaluronan Receptors/metabolism , Fibroblasts/drug effects
The effect of hydrazide linkers on the formation and mechanical properties of hyaluronan hydrogels was intensively evaluated. The reaction kinetics of hydrazone formation was monitored by NMR spectroscopy under physiological conditions where polyaldehyde hyaluronan (unsaturated: ΔHA-CHO, saturated: HA-CHO) was reacted with various hydrazides to form hydrogels. Linear (adipic, oxalic dihydrazide) and branched (N,N´,N´´-tris(hexanoylhydrazide-6-yl)phosphoric triamide and 4-arm-PEG hydrazide) hydrazides were compared as crosslinking agents. The mechanical properties of hydrogels were also modified by attaching a hydrophobic chain to HA-CHO; however, it was found that this modification did not lead to an increase in hydrogel stiffness. Cytotoxicity tests showed that all tested hydrazide crosslinkers reduced the viability of cells only slightly, and that the final hyaluronan hydrogels were non-toxic materials.
Cross-Linking Reagents/chemistry , Hyaluronic Acid/analogs & derivatives , Hydrazines/chemistry , Hydrazones/chemistry , Hydrogels/chemistry , Acylation , Animals , Biocompatible Materials/chemical synthesis , Biocompatible Materials/chemistry , Biocompatible Materials/toxicity , Cross-Linking Reagents/chemical synthesis , Cross-Linking Reagents/toxicity , Elastic Modulus , Hyaluronic Acid/chemical synthesis , Hyaluronic Acid/toxicity , Hydrazines/chemical synthesis , Hydrazines/toxicity , Hydrazones/chemical synthesis , Hydrazones/toxicity , Hydrogels/chemical synthesis , Hydrogels/toxicity , Hydrogen-Ion Concentration , Kinetics , Mice , Swiss 3T3 Cells
